穗花蛇菰雄花抽出物抗氧化及抗發炎成分之分離與鑑定

Abstract

穗花蛇菰為台灣傳統藥用作物，在民間常宣稱具有許多的療效。然而，目前對於穗花蛇菰中具生理活性之抽出成分，至今仍缺乏相關性研究。因此本試驗利用不同抗氧化評估模式，如：DPPH自由基清除能力試驗、超氧自由基清除能力試驗以及還原力試驗等，並配合總酚含量測定，以評估穗花蛇菰雄花及雌花抽出物之抗氧化活性及總酚含量差異。試驗結果顯示，穗花蛇菰雄花及雌花抽出物中，以雄花抽出物之抗氧化活性較佳。而利用液相–液相分配將雄花甲醇抽出物劃分為乙酸乙酯可溶部、正丁醇可溶部以及水可溶部後，則發現各可溶部中以乙酸乙酯可溶部抗氧化效果最佳。此外，穗花蛇菰雄花乙酸乙酯可溶部經管柱層析所得之10個次分離部（EA 1–EA 10）中，以EA 8具有最佳的DPPH自由基清除活性。而為進一步獲得EA 8中具抗氧化活性潛能之化合物，本研究續利用線上HPLC–DPPH試驗對其進行快速篩選，經分離及純化後共獲得1-O-(E)-Caffeoyl-β-D-glucopyranose（1）、1-O-(E)-p-Coumaroyl-β-D-glucopyranose（2）、Caffeic acid（3）、1,3-Di-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-β-D-glucopyranose（4）以及1-O-(E)-Caffeoyl-4,6-(S)-hexahydroxydiphenoyl-β-D-glucopyranose （5）5種具抗氧化潛力之化合物。而這些化合物中，以化合物1以及5具有較高的含量，於每克乙酸乙酯可溶部中其含量分別為11.5以及12.2 mg。此外，與常見之抗氧化物–兒茶素相較後發現，化合物4以及5較兒茶素具有更優異的DPPH自由基及超氧自由基清除能力。另一方面，在PMA誘導U937細胞產生促發炎細胞激素試驗方面，5種化合物中以化合物4具有最佳抗發炎能力，除可有效降低TNF–α及IL–1β之mRNA表現外，亦可抑制促發炎細胞激素之生成。綜合以上結果顯示，穗花蛇菰抽出物具有優異抗氧化及抗發炎活性，極具開發成為天然保健食品之潛能。

Balanophora laxiflora is a traditionally medicinal plant in Taiwan, and it is often claimed to be many curative effects. However, the potential bioactivities of B. laxiflora have not yet been studied completely to date. In this study, the antioxidant activities of the methanolic extracts from male and female flowers of B. laxiflora were evaluated by various in vitro antioxidant models, including DPPH radical scavenging assay, superoxide radical scavenging assay, and reducing power assay. In addition, the total phenolic contents were also assessed. Results revealed that, the methanolic extracts of male flowers of B. laxiflora had stronger antioxidant activities than those of female flowers. Furthermore, the methanolic extracts from the male flowers of B. laxiflora were fractionated to EtOAc, BuOH, and water soluble fractions by liquid-liquid partition. Of these, the EtOAc soluble fraction had the best antioxidant activities. Following by column chromatography, 10 subfractions (EA 1-EA 10) were obtained, and the EA 8 exhibited the strongest inhibitory activity against DPPH radical among them. Therefore, in order to rapidly screen the active phytochemicals from the EA 8, the online HPLC-DPPH method was used in this study. Five phytochemicals including 1-O-(E)-caffeoyl-β-D-glucopyranose (1), 1-O-(E)-p-coumaroyl-β-D-glucopyranose (2), caffeic acid (3), 1,3-di-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-β-D-glucopyranose (4), and 1-O-(E)-caffeoyl-4,6-(S)-hexahydroxydiphenoyl-β-D-glucopyranose (5) were isolated and identified. Of these, compounds 1 and 5 were found to be major bioactive compounds, and their contents were determined to be 11.5 mg and 12.2 mg per gramme of EtOAc fraction, respectively. Compared with (+)-catechin, a well-known antioxidant, compounds 4 and 5 exhibited stronger DPPH radical and superoxide radical scavenging activities than (+)-catechin. On the other hand, among 5 compounds, 1,3-di-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-β-D-glucopyranose (4) signifycantly inhibited TNF-α and IL-1β mRNA expression and cytokine production in PMA-stimulated U937 cells. These results demonstrate that the flower extracts of B. laxiflora have excellent antioxidant and antiinflammatory activities and thus have great potential as a source for natural health products.